Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, which are CLPP activators, which inhibit OXPHOS indirectly and have demonstrated safety in patients. METHODS: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201 and ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic efficacy in vivo in UM liver metastasis models. RESULTS: CLPP expression was detected in primary and mUM patient samples. ONC201 and 212 decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis in human UM cell lines in vitro. ONC212 inhibited OXPHOS, increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. CONCLUSIONS: Imipridones are a promising strategy for further testing and development in mUM.

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity.

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging.

Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the hetero-dimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.

ErbB protein modifications are secondary to severe cell membrane alterations induced by elisidepsin treatment.

Elisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points to the involvement of ErbB receptor tyrosine kinases and lipid rafts in the mechanism of action of elisidepsin, we investigated the effect of the drug on the distribution of ErbB proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing ErbB receptors. Stable expression of a single member of the ErbB family (ErbB1-3) or co-transfection of ErbB2 and ErbB3 did not modify the elisidepsin sensitivity of CHO and A431 cells. However, elisidepsin induced the redistribution of ErbB3 and two GPI-anchored proteins (transfected GPI-anchored eGFP and placental alkaline phosphatase) from the plasma membrane to intracellular vesicles without comparable effects on ErbB1 and ErbB2. Elisidepsin increased the binding of a conformational sensitive anti-ErbB3 antibody without modifying the binding of other ErbB2 or ErbB3 antibodies, and it decreased the homoassociation of both ErbB2 and ErbB3. We also found that elisidepsin decreased the fluorescence anisotropy of a membrane specific fluorescent probe and induced a blue shift in the emission spectrum of Laurdan pointing to significant changes in the order of the plasma membrane possibly associated with the formation of liquid ordered domains. Although the distribution of ErbB proteins is preferentially altered by elisidepsin, our data question their role in determining sensitivity to the drug. We assume that induction of liquid ordered domains is the primary action of elisidepsin leading to all the other observed changes.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and ß chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.

Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.

The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results.